WO2022143596A1

WO2022143596A1 - Surface display of antibodies in yeast cell

Info

Publication number: WO2022143596A1
Application number: PCT/CN2021/141864
Authority: WO
Inventors: Yiqi LIU; Man XU; Junqing MIAO; Deyuan DONG; Rong Li; Gennady Gololobov; Jijie Gu
Original assignee: Wuxi Biologics (Shanghai) Co., Ltd.; WuXi Biologics Ireland Limited
Priority date: 2020-12-29
Filing date: 2021-12-28
Publication date: 2022-07-07

Abstract

Provided is a method for displaying a polypeptide of interest such as full-length IgG antibody on yeast host cells, wherein heavy chain and light chain of polypeptide are expressed in two cassettes. ER-targeting peptide and the secretion signal peptide are linked to heavy chain and light chain separately. Also provided is a yeast host cells for expressing the polypeptide of interest on the surface of P. pastoris.

Description

Surface display of antibodies in yeast cell

Technical Field

The present invention relates to methods for displaying IgG antibody or libraries thereof on the surface of yeast host cells. The present invention provides expression vectors and helper display vectors which can be used in combinations for displaying polypeptides on the cell surface of yeast host cells. The display systems of the invention are particularly used for displaying a library of polypeptides.

Background of the Invention

Yeast display of antibody fragments has proven to be an efficient approach for novel antibody selection and engineering. Perhaps the biggest advantage of yeast display is its compatibility with fluorescent activated cell sorting (FACS) technology, which provides capability of separating antibody displayed cell repertoire into a purified fraction on the basis of fluorescence properties. Yeast display of proteins, especially antibody fragments scFv, was originally disclosed by Border and Wittrup (Boder, Eric T., and K. Dane Wittrup. "Yeast surface display for screening combinatorial polypeptide libraries. " Nature biotechnology 15.6 (1997) : 553-557) , they set up the platform links phenotype with genotype and provides general protocols of selecting high affinity scFv fragments through FACS technology. Over the last decade, yeast display has evolved dramatically, multiple antibody display formats have been implemented in yeast expression system. While the majority of these efforts focused on using antibody fragments as surrogates for lead identification rather than full-length immunoglobulin G proteins (IgGs) .

Yeast display of full-length IgG has also been reported with secretion and capture strategies, which relies on secretion of antibodies followed by capture to the surface by binding to a capturing agent. The researchers used biotinylated antibodies by extending the CH3 domain with a biotin ligase recognition sequence and co-expression of a biotin ligase in Saccharomyces cerevisiae (S. cerevisiae) followed by antibody capture to surface-immobilized avidin (Rakestraw, J.A., et al. "Secretion-and-capture cell-surface display for selection of target-binding proteins. " Protein Engineering, Design &Selection 24.6 (2011) : 525-530. ) . Another article described a dual-mode technique for engineering and production of full-length mAbs in Glyco-engineered Pichia pastoris (P. pastoris) . This published approach preserved co-secretion of full-length unmodified bivalent mAbs into culture medium while utilizing the natural antibody biogenesis pathway to covalently display monovalent functional half IgG molecules on the cell wall. They also described the first attempt to utilize this approach alongside haploid yeast mating by combining full-length heavy chain and full-length light chain libraries and the application of this display system to mature the expression of a monoclonal antibody lead while maintaining its affinity (Shaheen, Hussam H., et al. "A dual-mode surface display system for the maturation and production of monoclonal antibodies in glyco-engineered Pichia pastoris. " PloS one 8.7 (2013) . ) . A novel approach named REAL-Select was setup for the non-covalent display of IgG-molecules on the surface of yeast cells, which relies on the capture of secreted native full-length antibodies on the cell surface via binding to an externally immobilized ZZ domain, which tightly binds antibody Fc (Rhiel, Laura, et al. "REAL-Select: full-length antibody display and library screening by surface capture on yeast cells. " PloS one 9.12 (2014) . ) .

Yeast surface display of antibody fragments such as scFv or Fab formats have been implemented in many cases, while full-length antibody surface display in yeast were still on the trials. Full-length IgG display and selection technology may solve the mismatch problem which may happen in the IgG conversion process from selected scFv or Fab format leads, and the main obstacle may be the smaller library size since IgG display plasmid owns bigger size than scFv or Fab display vectors. Meanwhile, if the concentration of secreted soluble IgG is enough for functional screening directly, this technology will shorten the new drug molecules discovery period.

Summary of the Invention

One aspect of the present invention provides an expression vector, comprising polynucleotides encoding:

a) an antibody heavy chain expression cassette comprising: a promoter, an endoplasmic reticulum (ER) targeting peptide, an antibody heavy chain variable region (VH region) and an antibody heavy chain constant region; and

b) an antibody light chain expression cassette comprising: a promoter, a secretion signal peptide, an antibody light chain variable region (VL region) and an antibody light chain constant region.

In one embodiment of the present invention, the antibody heavy chain expression cassette further comprises a cell surface linker, preferably the cell surface linker is Aga2p, more preferably the Aga2p comprises: an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence which is at least 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 2.

In one embodiment of the present invention, Aga2p can be operationally linked at the C terminal of antibody heavy chain constant region by an amino acid sequence of SEQ ID NO: 3 ( (G ₄S) ₃) ; a tag such as c-Myc (EQKLISEEDL) can be operationally linked at the C terminal of antibody light chain constant region.

In one embodiment of the present invention, promoter is pAOX1 promoter.

In one embodiment of the present invention, the ER-targeting peptide comprises an amino acid sequence of SEQ ID NO: 4 (MQLLRCFSIFSVIASVLA) or an amino acid sequence which is at least 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 4.

In one embodiment of the present invention, the secretion signal peptide has an amino acid sequence of SEQ ID NO: 8 or an amino acid sequence which is at least 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 8.

In one embodiment of the present invention, the tag amino acid sequence is selected from the group of HA, c-Myc, His, and Flag tag. It should be understood by the skilled in the art that the peptide tag is not limited to the particular embodiments.

In one embodiment of the present invention, the antibody is a full-length IgG antibody. Preferably, the antibody is a full-length IgG antibody with a peptide tag.

Another aspect of the present invention provides a vector library comprising a plurality of vectors of present invention, each such vector comprises different VH region and/or VL region. In one embodiment, the vector library comprises at least 10^2, at least 10^3, at least 10^4, at least 10^6, at least 10^7, at least 10^8, at least 10^9 different vectors above.

Another aspect of the present invention provides an antibody display system comprising:

a) an isolated host cell;

b) one or more expression vectors of present invention; and

c) one or more polynucleotides encoding a cell surface anchoring protein.

In one embodiment of the present invention, the host cell is P. pastoris.

In one embodiment of the present invention, the cell surface anchoring protein is ScAga1 of SEQ ID NO: 1 or an amino acid sequence which is at least 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 1.

In one embodiment of the present invention, the system further comprises one or more polynucleotides encoding a molecular chaperone, preferably the molecular chaperon is PDI or BiP, more preferably, the PDI comprises an amino acid sequence of SEQ ID NO: 10 or an amino acid sequence which is at least 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 10; and the BiP comprises an amino acid sequence of SEQ ID NO: 11 or an amino acid sequence which is at least 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 11.

Another aspect of the present invention provides a method of displaying an antibody on a host cell, comprising the steps of:

a) introducing one or more expression vector of the present invention into the host cell;

b) introducing one or more polynucleotide encoding a cell surface anchoring protein into the host cell; and

c) culturing the host cell under condition whereby the polypeptide encoded by expression vector from a) and cell surface anchoring protein encoded by polynucleotide from b) are expressed.

In one embodiment of the present invention, the host cell is P. pastoris.

In one embodiment of the present invention, the method further comprising introducing one or more polynucleotides encoding a molecular chaperone, preferably the molecular chaperon is PDI or BiP, more preferably the PDI comprises an amino acid sequence of SEQ ID NO: 10 or an amino acid sequence which is at least 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 10; and the BiP comprises an amino acid sequence of SEQ ID NO: 11 or an amino acid sequence which is at least 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 11.

In one embodiment of the present invention, wherein the expression vector comprises polynucleotides encoding: an antibody heavy chain expression cassette comprising in order: a pAOX1 promoter, an ER-targeting peptide, a VH region, a heavy chain constant region, a linker, and an Aga2p; and an antibody light chain expression cassette comprising in order: a pAOX1 promoter, a secretion signal peptide, a VL region, an light chain constant region, and optionally a tag; wherein, the ER-targeting peptide is SEQ ID NO: 4, the secretion signal peptide is SEQ ID NO:8.

In one embodiment of the present invention, the polypeptide of interest is an antibody such as full-length IgG antibody or a library thereof. The antibody library comprises at least 10^2, at least 10^3, at least 10^4, at least 10^6, at least 10^7, at least 10^8, at least 10^9 different antibodies.

Brief Description of the Drawings

Figure 1 shows schematic diagram of full-length IgG display in P. pastoris. Cell surface anchored protein Aga1 from S. cerevisiae is heterologously expressed in P. pastoris, while heavy chain and light chain expression cassette are integrated into genome under the control of AOX1 promoter. Subunit of another yeast mating agglutinin Aga2 is fusion expressed at the carbon terminal of heavy chain, which can interact with Aga1 through a pair of disulfide bond. The three proteins are transferred to endoplasmic reticulum with the leading of ER-targeting peptide, in which molecular chaperones BiP and PDI facilitate the forming of inter-chain and intra-chain disulfide bonds. Consequently, a trimer complex containing heavy chain, light chain and ScAga1 are folded, and the complex is led by secretion signal peptide to Golgi apparatus to be further modified with glycosylation. Finally, full-length IgG is displayed on yeast cell surface through linkage between ScAga1 and ScAga2, and part of the expressed IgG are secreted to supernatant to form the soluble IgG.

Figure 2 shows cell surface anchor protein and full-length IgG display plasmids. AOX1 promoter is used to activate transcription of heterologous proteins, while general AOX1 transcription terminator is designed at the carbon terminal to compose an expression cassette. A) plasmid pPIC 3.5K is used to carry cell surface anchor protein amplified from S. cerevisiae; B) plasmid pPIC Z is used to carry full-length IgG display expression cassettes, double AOX1 promoter is employed to express heavy chain and light chain separately.

Figure 3 shows molecular chaperones overexpression plasmids. Plasmid pPIC 3.5K is used to carry endoplasmic reticulum molecular chaperones PDI and Bip. While gene aga1 and pdi are amplified from S. cerevisiae genome, gene bip from human is synthesized.

Figure 4 shows genome integration mechanism of IgG display vector in P. pastoris. Single recombination exchange is applied to integrate the IgG display vector into yeast genome, unique digestion site PmeⅠ is used to linearize the vector. After electroporation, the linearized vector will be integrated into yeast genome when genome DNA starts to replicate through homologous recombination.

Figure 5 shows function test result of cell surface anchor protein Sc_Aga1. Yeast agglutinin Aga2 is fused expressed with cMyc-tag, the plasmid is transformed into wild type with ScAga1 to construct strain WT-ScAga1&Aga2. Flow cytometer is used to test the interaction function of ScAga1 and ScAga2.

Figure 6 shows schematic diagram of leading peptide optimization strategies. Three combination of secretion signal peptide (SSp) with endoplasmic reticulum peptide (ERp) are constructed in pPIC Z vector.

Figure 7 shows results of leading peptide optimization strategy. Flow cytometry is used to test the cell surface expression and antigen binding, yeast strains without IgG display vector were tested in parallel as the negative control. Comparisons between different leading peptide combined strategies. SSp-H&SSp-L means both heavy chain (H) and light chain (L) are led by secretion signal peptide (SSp) , ERp-H&SSp-L means heavy chain (H) is led by secretion signal peptide (SSp) , while light chain is led by endoplasmic reticulum peptide (ERp) , ERp-H&SSp-L means both heavy chain (H) and light chain (L) are led by endoplasmic reticulum peptide (ERp) .

Figure 8 shows supernatant soluble IgG expression and antigen-binding comparison between SSp-H&L and ERp-H&SSp-L strains. Supernatant ELISA is tested to determine the expression level A) and antigen binding level B) .

Figure 9 shows supernatant soluble IgG expression and antigen-binding comparison between ERp-H&L and ERp-H&SSp-L strains. Supernatant ELISA is tested to determine the expression level A) and antigen binding level B) .

Figure 10 shows results of protein purification of soluble IgG from supernatant. Reducing and non-reducing conditions are used to treat the samples to isolate heavy chain and light chain protein fragments.

Figure 11 shows functional assay of yeast expressed IgG. A) Binding curve of yeast secreted IgG with hPro1 displayed cell was measured in gradient dilution, stain Pp_Aga1 without IgG display vector was tested as negative control, while uIgG1K was tested as positive control. B) Activation curve of yeast secreted IgG with Jurkat hPro1 cell was measured in gradient dilution, yeast medium (BMMY) and mammalian cell medium (RPM1 1640) were used to dilute the purified soluble IgG.

Detailed description of the Invention

The following description of the disclosure is merely intended to illustrate various embodiments of the disclosure. As such, the specific modifications discussed are not to be construed as limitations on the scope of the disclosure. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the disclosure, and it is understood that such equivalent embodiments are to be included herein. All references cited herein, including publications, patents and patent applications are incorporated herein by reference in their entirety.

Definitions

The articles “a” , “an” , and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “a polypeptide complex” means one polypeptide complex or more than one polypeptide complex.

Throughout this disclosure, unless the context requires otherwise, the words “comprise” , “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of” . Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

The terms “polypeptide” , “peptide” , and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, or an assembly of multiple polymers of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. An alpha-carbon refers to the first carbon atom that attaches to a functional group, such as a carbonyl. A beta-carbon refers to the second carbon atom linked to the alpha-carbon, and the system continues naming the carbons in alphabetical order with Greek letters. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The term “protein” typically refers to large polypeptides. The term “peptide” typically refers to short polypeptides. Polypeptide sequences are usually described as the left-hand end of a polypeptide sequence is the amino-terminus (N-terminus) ; the right-hand end of a polypeptide sequence is the carboxyl-terminus (C-terminus) . “Polypeptide complex” as used herein refers to a complex comprising one or more polypeptides that are associated to perform certain functions. In certain embodiments, the polypeptides are immune-related such as antibody.

The term “antibody” as referred to herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion" ) or single chains thereof. An "antibody" refers to a protein comprising at least two heavy (H) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. The VH regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) , interspersed with regions that are more conserved, termed framework regions (FR) . Each VH is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy chains contain a binding domain that interacts with an antigen. The CDRs in heavy chain are abbreviated as H-CDRs, for example H-CDR1, H-CDR2, H-CDR3. An "antibody" also refers to a protein comprising at least two light (L) chains comprised of three domains, L-CH1, L-CH2 and L-CH3.

The term "antibody" as used in this disclosure, refers to an immunoglobulin or a fragment or a derivative thereof, and encompasses any polypeptide comprising an antigen-binding site, regardless whether it is produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies.

The term "IgG antibody" is full-length IgG comprising at least two heavy (H) chains inter-connected by disulfide bonds and at least two light (L) chains, in which the heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (CH) . The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3 domain. The light (L) chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL) .

“CH2 domain” as used herein refers to includes the portion of a heavy chain molecule that extends, e.g., from about amino acid 244 to amino acid 360 of an IgG antibody using conventional numbering schemes (amino acids 244 to 360, Kabat numbering system; and amino acids 231-340, EU numbering system; see Kabat, E., et al., U.S. Department of Health and Human Services, (1983) ) .

The “CH3 domain” extends from the CH2 domain to the C-terminus of the IgG molecule and comprises approximately 108 amino acids. Certain immunoglobulin classes, e.g., IgM, further include a CH4 region.

“Percent (%) identical to” with respect to amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical amino acids (or nucleic acids) . Conservative substitution of the amino acid residues may or may not be considered as identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI) , see also, Altschul S.F. et al., J. Mol. Biol., 215: 403-410 (1990) ; Stephen F. et al., Nucleic Acids Res., 25: 3389-3402 (1997) ) , ClustalW2 (available on the website of European Bioinformatics Institute, see also, Higgins D.G. et al., Methods in Enzymology, 266: 383-402 (1996) ; Larkin M. A. et al., Bioinformatics (Oxford, England) , 23 (21) : 2947-8 (2007) ) , and ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by the tool, or may customize the parameters as appropriate for the alignment, such as for example, by selecting a suitable algorithm.

The term “binding” or “binds” as used herein refers to a non-random binding reaction between two molecules, such as for example between an antibody and an antigen. In certain embodiments, the polypeptide complex and the bispecific polypeptide complex provided herein specifically bind an antigen with a binding affinity (K _D) of ≤ 10 ^-6 M (e.g., ≤ 5×10 ^-7 M, ≤ 2×10 ^-7 M, ≤ 10 ^-7 M, ≤ 5×10 ^-8 M, ≤2×10 ^-8 M, ≤ 10 ^-8 M, ≤ 5×10 ^-9 M, ≤ 2×10 ^-9 M, ≤ 10 ^-9 M, or ≤ 10 ^-10 M) . K _D as used herein refers to the ratio of the dissociation rate to the association rate (k off/k on) , may be determined using surface plasmon resonance methods for example using instrument such as Biacore.

The term “nucleic acid” or “polynucleotide” as used herein refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses polynucleotides containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) , alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19: 5081 (1991) ; Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985) ; and Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994) ) .

The encoding polynucleotide sequences can be further operably linked to one or more regulatory sequences, optionally in an expression vector, such that the expression or production of the first and the second polypeptides is feasible and under proper control.

The encoding polynucleotide sequence (s) can be inserted into a vector for further cloning (amplification of the DNA) or for expression, using recombinant techniques known in the art. Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

The term “vector” as used herein refers to a vehicle into which a polynucleotide encoding a protein may be operably inserted so as to bring about the expression of that protein. Typically, the construct also includes appropriate regulatory sequences. For example, the polynucleotide molecule can include regulatory sequences located in the 5’-flanking region of the nucleotide sequence encoding the guide RNA and/or the nucleotide sequence encoding a site-directed modifying polypeptide, operably linked to the coding sequences in a manner capable of expressing the desired transcript/gene in a host cell. A vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of the genetic element it carries within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) , or P1-derived artificial chromosome (PAC) , bacteriophages such as lambda phage or M13 phage, and animal viruses. Categories of animal viruses used as vectors include retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40) . A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. A vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating.

The phrase “host cell” as used herein refers to a cell into which an exogenous polynucleotide and/or a vector has been introduced. Suitable host cells for cloning or expressing the DNA in the vectors herein are yeast cells.

Host cells are transformed with the above-described expression or cloning vectors can be cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the cloning vectors.

Examples

Example 1: Illustration of yeast full-length IgG display system

Theoretical design of yeast full-length IgG display system is described in this example (Figure 1) .

Pichia pastoris (P. pastoris) is used as the host to display full-length IgG. Cell surface anchored protein Aga1 from Saccharomyces cerevisiae (ScAga1, SEQ ID NO: 1) was heterologously expressed in P. pastoris, while subunit of Aga2 (ScAga2 subunit, SEQ ID NO: 2) was fused at the carbon terminal of heavy chain fragment of IgG. Light chain fragment was expressed via same vector with heavy chain, but separate express cassette. As a result, translated antibody fragments and cell anchor protein were transferred into endoplasmic reticulum to be assembled, molecular chaperones facilitated forming of inter-chain and intra-chain disulfide bonds. Pro-region in the secretion signal peptide led the antibody-ScAga1 complex to Golgi apparatus to be further modified by glycosylation, then the complex was secreted out of cell through secretory vesicles. During secretion process, heavy chain fragment was tethered to ScAga1 via Aga2-Aga1 interaction. Since ScAga1 is uniformly distributed on yeast surface, full-length IgG will be displayed on surface consequently. When yeast expressed IgG is saturated on surface, the redundant IgG will be secreted into supernatant to form soluble IgG.

Promoters play vital roles in the transcript process, natural alcohol oxidase 1 promoter (pAOX1) was used to express heavy chain, light chain and cell surface anchor protein. The display vector is integrated into yeast genome through homologous recombination, restrict digestion site Pme1 in pAOX1 promoter is used to linearize the vector to help improve the integration efficiency. Molecular chaperones function to facilitate the folding of heavy chain and light chain, and overexpression of BiP and/or PDI showed a certain degree of positive effects. Secretion signal peptide contains two main regions, pre-region and pro-region. Pre-region leads the proteins to endoplasmic reticulum, while pro-region functions to transfer proteins from endoplasmic reticulum to Golgi apparatus. Retention time of antibody fragments in endoplasmic reticulum is related to the strength of secretion signal peptide, therefore trials on different combinations of leading peptide in heavy chain and light chain may enhance the antibody assemble effectiveness and efficiency. Soluble full-length IgG in supernatant is usually used for screening, concentration of soluble IgG determines what levels of screening method can be done. Functional screening needs higher concentration of soluble IgG than simple ELISA screening, so IgG expression level is one of the vital evaluation indexes should be considered. Detailed designs and optimizations are listed in the following examples.

Example 2: Full-length IgG display vectors construction

Yeast cell surface anchored protein and full-length IgG display vectors construction process are described in this example.

As shown in Figure 2, cell surface anchor protein ScAga1 (SEQ ID NO: 1) from S. cerevisiae was constructed into P. pastoris expression vector to get plasmid pPIC 3.5K_ScAga1, the construction process was as follows. Fragment ScAga1 was amplified using primers BamH～ScAga1-F and EcoR～ScAga1-R with purified S. cerevisiae genome as the PCR template, while P. pastoris plasmid pPIC3.5K was digested with BamH1 and EcoR1, both the PCR fragment and digested vector were gel isolated and purified with correct size. The target PCR fragment and digested vector were then incubated with seamless cloning kit and transformed into TG1 competent cell. The transformants were recovered with LLB medium at 37℃ for 1 h, then the culture was plated on LLB plates with Zeocin as the selection press. Colony PCR was conducted with primers 5’AOX1-F and 3’AOX1-R to identify single clones harboring integrated plasmid. Finally, the constructed plasmids were sent for sequencing to align with template sequence to further confirm the plasmid. The constructed plasmid pPIC3.5K_ScAga1 could be linearized with Pme1, and integrated into genome with the AOX1 promoter as the homologous recombination site. As a result, the yeast transformants carrying plasmid pPIC3.5K_ScAga1 express cell surface anchored protein at cell surface.

Plasmid construction process of the full-length IgG display plasmid was described as follows. Heavy chain constant region (e.g. IgG1 or IgG4_S228P) was designed to be ligated with subunit of ScAga2 (SEQ ID NO: 2) through linker (SEQ ID NO: 3) , and the expression cassette was led by ER-targeting peptide (SEQ ID NO: 4) , the sequences of the components were shown in SEQ ID NOs: 4, 9 or 5, 3, 2. SEQ ID NO: 5 is IgG4_S228P constant region and SEQ ID NO: 9 is IgG constant region. While light chain expression cassette was designed to be led by secretion signal peptide (SEQ ID NO: 8) , and the sequences of light chain constant region were listed in SEQ ID NOs: 6-7. The heavy chain and light chain expression cassette were synthesized according to the sequence listing.

A unique digestion site BamH1 was designed at the outside flanking region of terminator, which was used to linearize the plasmid harboring heavy chain expression cassette. Light chain expression cassette was amplified by PCR and isolated through gel electrophoresis, the expression cassette was inserted into heavy chain expression plasmid to get the plasmid containing both heavy chain and light chain expression cassette. Finally, the constructed plasmids were sent for sequencing to align with template sequence to further confirm the plasmid. The constructed full-length IgG display plasmid was linearized with Pme1, and integrated into yeast genome regarding AOX1 promoter as the homologous recombination site.

Example 3: Construction of molecular chaperones overexpression plasmids

Construction of molecular chaperones overexpression plasmids is described in this example.

The chaperone protein disulfide isomerase (PDI) , as shown in SEQ ID NO: 10, was reported to be beneficial to scFv expression in yeast, while another chaperone BiP (SEQ ID NO: 11) , a member of the Hsp70 chaperone family, plays a crucial role in CH1 inter-chain disulfide bond formation. Plasmid pPIC 3.5K was used to conduct the host engineering modification, based on cell surface anchor Aga1 expression plasmid, PDI overexpression cassette was inserted closely to the Aga1 expression cassette. Constitutive promoter pGAPH was used to activate the transcription of PDI and BiP, and promoter fragments were amplified from yeast genome through PCR. PDI and BiP expression cassettes were spliced separately using seamless cloning kit, followed by assembling the expression cassettes into one plasmid. Consequently, plasmids pPIC 3.5K_ScAga1+PDI, pPIC 3.5K+ScAga1+BiP and pPIC 3.5K_ScAga1+PDI+BiP were obtained (Figure 3) . Single digestion site Pme1 was retained at each vector to linearize and integrate into yeast genome. Human-derived BiP was truncated to remove its original signal peptide, and replaced with yeast endoplasmic reticulum leading peptide to ensure that hybrid BiP will function at endoplasmic reticulum.

Example 4: Linearization and genome integration in yeast

As shown in Figure 4, the linearization and genome integration mechanism in yeast is described in this example.

The pAOX1 promoter in yeast expression vector shares same sequence with the pAOX1 promoter in yeast genome, and it can be linearized by single site Pme1 to expose homologous flanks at both N-terminal and C-terminal of the vector. The linearized vector is transferred into yeast cell by electroporation, and it will be integrated into yeast genome when it is replicating. As a result, all the vector components will be integrated into yeast genome, including the selection marker Zeocin expression cassette, replicate origins etcetera. And hybrid pAOX1 promoters will be formed, which will not have influence on expression of alcohol oxidase enzyme, and methanol consuming efficiency will be kept normal with wild type yeast. Since linearization and electroporation of target plasmid forms hybrid expression cassette, so linearization site on the plasmid should be carefully considered to avoid frame shift integration. Digestion site Pme1 in pAOX1 promoter is a unique digestion site in IgG display vector so that it can be commonly used in integration process.

Example 5: Functional test of the cell anchored protein amplified from S. cerevisiae

Functional test of the cell anchored protein amplified from S. cerevisiae is described in this example.

Yeast cell surface anchored protein gene Sc_Aga1 was heterologously expressed using vector pPIC 3.5K, while subunit of Sc_Aga2 was co-expressed with Sc_Aga1 using another selective plasmid pPIC Z. A protein tag cMyc was fused expressed at the carbon terminal of ScAga2 as a labeling for cell cytometry test. In this example, plasmids pPIC 3.5K_ScAga1 and pPIC Z_ScAga2 were co-transferred into wild type yeast, double selective plate was used to select positive transformants. After induced by methanol for 48 hours, the strains were washed with PBSA buffer and labeled with anti-cMyc PE to test the function of ScAga1 and ScAga2. As shown in Figure 4, wild type strains showed no PE signal while yeast transformants with ScAga1 and ScAga2 showed a stronger fluorescence intensity. The results stated that cell surface anchored protein ScAga1 with its partner subunit ScAga2 worked well in P. pastoris, and both of the two proteins will be used to display full-length antibody on yeast surface.

Example 6: Full-length antibody displayed vector and vector library

Full-length antibody displayed vector design and leading peptide optimization strategies are described in this example.

Full-length IgG contains two pair of heavy chain and light chain, here double promoter (pAOX1 promoter) was used to activate the transcription of heavy chain and light chain respectively. To keep the linearization site be the unique, digestion site, Pme1 at the light chain promoter was mutated to retain the heavy chain’s Pme1 as the only digestion site in the entire vector. Leading sequence is essential on antibody expression in yeast. Three combinations of secretion signal peptide with endoplasmic reticulum peptide were designed. For short, SSp is short of secretion signal peptide and ERp is short of endoplasmic reticulum peptide (ER Targeting peptide) . Combinations such as SSp-H&Lchain, SSp-Lchain&ERp-Hchain and ERp-H&Lchain were designed and constructed (Figure 6) . Considering that expression level of heavy chain is usually lower than that of light chain fragment because of their different molecular weight, these combinations of leading peptides will help enhance the antibody folding and transport in yeast cell.

Example 7: surface display of full-length IgG using methods

This example illustrates surface display of full-length IgG (Sainson, R.C.A., Arkinstall, S.J., Campbell, J. I., Ali, M.H., Lee, E.C., McCourt, M.J., ... &Kosmac, M. (2018) . U.S. Patent No. 9,957,323. Washington, DC: U.S. Patent and Trademark Office) using methods disclosed herein.

Flow cytometry was used to test the surface display of full-length IgGs, light chain fragment was expressed with a c-Myc tag at carbon terminal so that full-length IgG cell surface expression can be detected with fluorescent dye anti-cMyc-PE. While biotinylated antigen was incubated with induced yeast cell and then labeled with Streptavidin-Alex 647 to test specific antigen binding display. Display test was conducted as follows, antibody display vector was linearized with Pme1 and transformed into yeast competent cell which harboring cell surface anchored protein ScAga1, then plated on YPD (Yeast Extract Peptone Dextrose Medium) with Zeocin medium. After cultivation at 30℃ for 3 days, single yeast colony was picked into BMMY medium, and incubated at 30℃ for 72h, while during induction process, 1%methanol was supplemented in every 24 h. Finally, 200 μL induced medium was collected and centrifuged at 6000 rpm, 4℃ for 3min to get cell pellet. Ice-cold PBSA buffer was used to wash the yeast cells twice. Biotinylated antigen was incubated with washed yeast cells, at least 25 nM of biotinylated antigen should be used, at room temperature for 1h. Ice-cold PBSA buffer was used to wash the cells and remove the non-binding antigens, then cell pellet was resuspended with 200 μL PBSA buffer. Fluorescent dyes streptavidin Alex 647 and anti-cMyc-PE were incubated with the ratio of 1: 200 at 4℃ for 1 h, then washed with ice-cold PBSA buffer and detected with flow cytometry. As shown in Figure 7, double stain method was used and wild type strain was treated with same conditions as a negative control. Yeast strains with SSp-H&SSp-L showed approximate 14.2%cell populations having both full-length antibody surface expression and specific antigen binding, the positive cell percentage was much lower than the yeast transformants with ERp-H&SSp-L or ERp-H&L.

Endoplasmic reticulum leading peptide plays a vital role on increasing endoplasmic reticulum retention time, as a result, the antibody fragments led by ERp will have more probabilities to function with molecular chaperones. But this function will decrease the transport efficiency from endoplasmic reticulum to Golgi apparatus. Replacing heavy chain’s leading peptide from secretion signal peptide to endoplasmic reticulum peptide increased the double positive cells’ population to 93.6% (Figure 7) , while the same changes on light chain did not get a significant improve based on ERp-H&SSp-L strains. To select optimal combination of the secretion signal peptide and endoplasmic reticulum peptide, more evaluations such as supernatant soluble IgG concentration were needed. In conclusion, endoplasmic reticulum peptide on heavy chain benefits full-length IgG display on yeast surface.

Example 8: Soluble full-length antibody concentration in supernatant

Detection of soluble full-length antibody concentration in supernatant is described in this example.

Yeast expressed antibodies are secreted bypass the cell wall, during this process, the cell anchored proteins on yeast surface will be saturated if the yield is much higher than ScAga1 numbers on yeast surface. Expression level and antigen binding ability were tested using ELISA, the experiments were conducted as follows.

To test the expression level at supernatant, anti-hFc antibody with 1 μg/mL concentration was coated on ELISA plates at 4℃ overnight, washed the plate with 1×PBST and added 200 μL blocking buffer to each well and incubated the plate at 25℃ for 1h, followed by washing the plate with 1×PBST 3 times. After induction, 100 μL of supernatant was diluted with 2 times using casein buffer as the initial testing sample, then the samples were serially diluted with 3 times and loaded in the plate wells at 25℃ for 1h, followed by washing the plate with 1×PBST for 3 times. Secondary antibody anti-human Kappa Constant-HRP (1: 4000 diluted) was used and incubate the ELISA plate at 25℃ for 1 h with shaking. Washed the plate with 1×PBST for six times, then added 100 μL of substrate mixture (1: 1 dilution of substrate A and B from TMB) to each well, incubated at 25℃ in dark for 10 min. Added 100 μL of 2M HCl to each well to stop the reaction and red the absorbance values at 450 nm.

To test antigen binding value of the soluble IgG in supernatant, capture ELISA was conducted. Anti-human Fc antibody with concentration of 1μg/mL was used to coat on ELISA plates at 4℃ overnight, followed by washing the plate with 1×PBST for one time. Added 200 μL blocking buffer to each well and incubate the plate at 25℃ for 1h, followed by washing the plate with 1×PBST for 3 times. Supernatant with volume 100 μL of was diluted for two times with casein and added into each well, incubated the plates at 25℃ for 1 h with shaking. Washed the plate with 1×PBST for 3 times and added 1 μg/mL biotinylated antigen at 25℃ for 1 h, followed by washing the plate with 1×PBST for 3 times. Added 100 μL SA-HRP (1:20000) to each well of the plate and incubated at 25℃ for 1 h, followed by washing with 1×PBST for 6 times. Substrate mixture (1: 1 dilution of substrate A and B from TMB) was added to each well, and incubated the plate at 25℃ in dark for10 min. Finally, 100 μL of 2M HCl was added to each well to stop the reaction and red the absorbance value at 450 nm.

As shown in Figure 8, strains with ERp-H&SSp-L display vectors expressed higher amount of soluble IgG into supernatant, compared with strains with SSp-H&L, about 6-fold increase. While the supernatant from the two types of strains did not show obvious difference on soluble IgG binding with antigen. Supernatant from wild type or medium did not show positive signals. As shown in Figure 9, soluble IgG concentration from strains transformed with ERp-H&L were lower than strains transformed with ERp-H&SSp-L, while the antigen binding ability showed on difference. Taking the soluble expression level as an evaluation criterion, vector with combination of ERp-H&SSp-L behaved better than other vectors.

Taking the expression and capture ELISA results into consideration, vector combining with ERp-H&SSp-L was the optimal display vector to achieve both cell surface display and soluble IgG expression. Consequently, heavy chain leading by endoplasmic reticulum peptide and light chain leading by secretion signal peptide was the optimized combination.

Example 9: Full-length IgG purification and concentration determination

Full-length IgG purification and concentration determination are demonstrated in this example.

Soluble IgG will be formed in the supernatant when cell surface anchor proteins were saturated, ELISA experiments were conducted to validate the viewpoint. To determine the supernatant concentration precisely, purification through protein A column was conducted followed by purity and concentration test. Yeast transformants were firstly detected by flow cytometry to ensure full-length IgG has been displayed on the yeast surface, then the strains were recovered in MGY medium with Zeocin till it reached exponential phase. Subsequently, the strains were collected by centrifuge at 4℃ and resuspened with BMMY medium at an initial OD=0.5, then the strains were cultivated at 30℃ for 72 h. Supernatant was obtained by centrifuge, and approximate 150 mL supernatant was used to load on protein A column to purify soluble IgG. Finally, 100 μL elution buffer was used to dissolve, and the purified samples were detected in the SDS-page with supernatant samples, after treating at reducing and non-reducing conditions. As shown in Figure 10, under non-reducing conditions, both supernatant samples and purified samples showed a band at 150 kD, which is consistent with theoretical IgG molecular weight size. The purification process had concentrated the target protein by comparing the brightness of bands of supernatant and purified samples. When treated the samples with denaturing reagents, other two bands with size of 65 kD and 28 kD respectively were observed, which should be the isolated heavy chain and light chain. These data proved that soluble IgG formed in the supernatant, and it can be concentrated and purified by protein A column. Purity of the samples were determined by analyze the brightness of the target band and unrelated bands, results showed that around 70%of total proteins were full-length IgG. And the protein concentration was detected using ultraviolent absorption apparatus, the concentrated value was 0.47±0.03 mg/mL. Yield of strains was calculated to be 0.18±0.01 mg/L, which was enough for the simple ELISA screening, but for functional screening the expression capability should be further enhanced.

Example 10: in vitro functional characterization of yeast expressed IgG

This example demonstrates the in vitro functional characterization of yeast expressed IgG.

Since yeast has different post-translational modification compared with humans, especially the glycosylation process. To test whether yeast expressed IgG can be directly used for functional screening, the supernatant from IgG displayed strains was purified and detected with in vitro functional characterization. Target antigen was named hPro1 and expressed at cell surface of 293 cells, while purified antibodies from yeast supernatant were tested in parallel with mammalian expressed IgG, named IgG-PC. The samples were normalized to 100 nM and serially diluted with 5-fold using 1%BSA buffer or yeast induction BMY medium, and each well were incubated with 10 ⁵ hPro1 displayed cells at 4℃ for 1 h. Secondary antibody goat anti-human IgG Fc-Alexa647 was diluted with 1: 500 at 1%BSA buffer, each well was incubated with 100 μL diluted secondary antibody at 4℃ for 0.5 h, followed by centrifuge to discard the supernatant and resuspened with 80 μL 4%paraformaldehyde (PFA) . The samples were analyzed by flow cytometry, and the results were shown in Figure 11. Wild type strains transformed with ScAga1 only (Aga1) was analyzed as a negative control, while another purified IgG was tested as the negative control (IgG-NC) to the hPro1 displayed cells, both of the two negative samples showed on mean fluorescence intensity. The results demonstrated that the functional assay was set up well, which can be used for the functional assay of yeast supernatant. A purified benchmark IgG antibody was tested in parallel as a positive control, named IgG-PC, which has the function of activation of transfected Jurkat cells. Yeast supernatants diluted with 1%BSA and BMY medium showed strong binding intensity with hPro1 cells, as shown in table 1, the maximum MFI of the two samples reached 40800±5100 at 1%BSA buffer and 41250±2850 at BMY medium respectively. As for EC50, yeast expressed IgG behaved at similar level compared to mammalian cell expressed IgG. The results demonstrated that yeast expressed IgG had comparable antigen binding ability with mammalian cells, and the yeast induction medium BMY had no impact on the assay, therefore yeast expressed soluble IgG can be directly used for functional assay if the concentration is higher than around 10 nM.

Jurkat cells transfected with antigen activation pathways were used to test the function of yeast expressed IgG. Anti-human-Fc antibody was dissolved in DPBS buffer to 200 nM, which functioned as the cross-linker to fix samples on plates, the coating process was maintained at 4℃ overnight. Then the IgG samples were serially diluted with 3-fold using mammalian medium RPMI1640 or yeast medium BMY, and incubated at 37℃ for 5 h. Fluorescence substrate was added into the reaction system, then incubated at 30℃ for 10 min followed by intensity detection on Envision. Results were illustrated in Figure 11, and the values were listed in table 2. Two yeast expressed samples were tested, the maximum value was 57940±5860 at mammalian cell medium while it decreased to 27900±8380 at yeast medium. The values of positive control sample showed similar tendency that reduced from 84520 at mammalian cell medium to 15160 at yeast medium. Considering that methanol is the key component in yeast medium, which functions as the inducer for IgG transcription, might be toxic to the Jurkat cell. It might be the reason why signal intensity dropped at yeast medium, but taking the signal of negative control in yeast medium into consideration, it created a broader detection window from 960 to 36280 than mammalian cell medium. As for EC50, yeast samples showed comparable value with positive control, which demonstrated that the yeast expressed functional IgG. In conclusion, soluble IgG from yeast displayed strains showed normal functionality of antigen binding and Jurkat cell activation. To directly use supernatant for functional screening, the IgG yield in yeast should be further enhance to at least 10 nM.

Table 1

Table 2

Example 11: Surface display of full-length IgG library and selection of antibody of interest

Generation of yeast IgG library from phage scFv selection outputs, or synthetic DNA library were described in this example.

After three rounds of phage panning, the library diversity will be usually enriched from 1E+12 to 1E+06, which is equivalent to an achievable yeast library size. Consequently, the scFv outputs from phage panning was designed to be batch reformatted t to full-length IgG library in yeast, followed by functional selection based on IgG format. This design combines phage library’s advantages, such as big library size and high diversity, with yeast IgG library’s strength on full-length IgG surface display and real-time selection on flow cytometer.

Plasmids DNA were prepared from selected scFv phage display library for use as PCR template using standard methods. PCR reactions were conducted to amplify the VH-VL paired scFv, and seamless cloning kit was used to insert scFv DNA fragments into yeast display vector, which was digested with EcoRⅠ and XbaⅠ. Full-length IgG constant region and yeast promoter was amplified by another PCR reactions and purified by Qiagen gel isolation kit, this fragment was cloned into scFv inserted yeast display vector using seamless cloning kit. Considering that the selected scFv plasmids were a repertoire with 1E+06 diversity, primers used for scFv amplification should contain all the human resource antibody genes. Consequently, the primers were designed according to the disclosed antibody gene sequences from IMGT database, and the primers should cover all the disclosed human antibody genes. Through two-step cloning method, the pre-selected scFv plasmids were batch reformatted into yeast IgG display vectors. The reformatting throughput can be achieved with a range around 1E+07, which is ten-fold higher than theoretical diversity 1E+06. To control the quality of two-step cloning, library size titration, colony PCR and clone sequencing were needed, usually 10-fold the theoretical diversity should be achieved for library size and correct rate from sequencing should be consistent with the selected phage expression rate. Quality fulfilled libraries were collected and used to extract plasmid libraries through plasmid extraction kit.

To construct yeast IgG display library, the IgG display vectors should be linearized to be integrated into yeast genome by homologous recombination. Digestion site PmeⅠ in the middle region of pAOX1 promoter was retained to linearize IgG display plasmids. Yeast strain containing Sc_Aga1 expression cassette serves as the host and it was used to make yeast competent cells with regular yeast competent cell preparation protocol. The linearized IgG display vectors were gently mixed with yeast competent cells, and equally divided into electroporation cup, 4 μg linearized vector mixed with 400 μg yeast competent cell. The electroporation was conducted under 2.5 kV, pre-warmed medium should be added into electroporation cup immediately after pulsing. Collected all the electroporation culture into 50 mL cone tube, and recovered at 30 ℃ with MGYZ medium overnight to passage one to two times. At the same time, the recovered library was serially diluted and plated on MGYZ plates, cultured at 30 ℃ for 2 to 3 days, to estimate library size. The isolated yeast colonies were sent for sequencing to identify the antibody sequences, while correct rate and library diversity were evaluated according to the sequence analysis data. A yeast library, with amenable library size 10-fold covering theoretical diversity and good correct rate determined well-integrated IgG display plasmid into genome, will move to selection process of specific antigen binding sequences.

Selection of yeast IgG display library by magnetic beads sorting was described as follows. Recovered the stocked yeast library at 30℃ with shaking overnight till middle-log phase, then cultured the cells for another passage to OD=1. Pellet yeast cell with a population 10-fold higher than theoretical library size by centrifuge, followed by resuspension with methanol induce medium and induction for 72 h at 30℃. Induced yeast cells were washed with ice-cold PBS for 2 times, and incubated with biotinylated antigen at 30℃ for 30 min, the antigen concentration should be adjusted according to different needs. Followed by centrifuge at 4℃ with 10000 rpm for 2 min, and the cell pellet was washed with ice-cold PBS for 2 times. Meanwhile, magnetic beads were washed with ice-cold PBS for 2 times and incubated with antigen-incubated yeast cells for 1 h at 30℃ with rotation. Then transferred the mixture to magnetic shelf for 5 min till the beads were collected into the bottom of tube. Removed the supernatant and washed with ice-cold PBS buffer for 2 times with rotation, the cell-beads complex was inoculated into MGY medium and cultured at 30℃ overnight. The magnetic beads were removed by placing at magnetic shelf for 5 min, the yeast cells were re-induced at BMMY medium for 72 h for the next round of magnetic beads sorting or FACS sorting.

Selection of yeast IgG display library by FACS sorting was described as follows. Induced yeast cells were washed with PBS buffer, and incubated with biotinylated antigen at 30℃ for 30 min. Secondary antibody anti-cMyc-PE and SA-Alexa647 were incubated with yeast cells on ice followed by washing with PBS buffer. The washed yeast cells were purified using 40 μm filter to remove the cell inclusions and sampled into FACS system, the sorting gate was set to select double-positive populations. After several rounds of FACS sorting, top clones were isolated by MGY plates and induced at BMMY medium for 72 h. The supernatant was detected by ELISA to identify high affinity clones, while the strains were sent for sequencing to identify the clone sequence.

Claims

An expression vector, comprising polynucleotides encoding:

a) an antibody heavy chain expression cassette comprising: a promoter, an endoplasmic reticulum (ER) targeting peptide, a heavy chain variable region (VH region) and a heavy chain constant region; and

b) an antibody light chain expression cassette comprising: a promoter, a secretion signal peptide, a light chain variable region (VL region) and a light chain constant region.
The vector of claim 1, wherein the antibody heavy chain expression cassette further comprises a cell surface linker, preferably the cell surface linker is Aga2p, more preferably the Aga2p comprises:

(a) an amino acid sequence of SEQ ID NO: 2; or

(b) an amino acid sequence which is at least 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 2.
The vector of claim 1, wherein the promoter is pAOX1 promoter.
The vector of claim 1, wherein the ER-targeting peptide comprises:

(a) an amino acid sequence of SEQ ID NO: 4; or

(b) an amino acid sequence which is at least 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 4.
The vector of claim 1, wherein the secretion signal peptide comprises:

(a) an amino acid sequence of SEQ ID NO: 8; or

(b) an amino acid sequence which is at least 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 8.
The vector of claim 1, wherein the antibody is a full-length IgG antibody, preferably the antibody is full-length IgG antibody labeled with a peptide tag.
A vector library comprising a plurality of vectors according to any one of claims 1-6, wherein each such vector comprises different VH region and/or VL region.
An antibody display system comprising:

a) an isolated host cell;

b) one or more expression vectors of any one of claims 1-6; and

c) one or more polynucleotides encoding a cell surface anchoring protein.
The system of claim 8, wherein the host cell is P. pastoris.
The system of claim 8, wherein the cell surface anchoring protein is ScAga1 comprising:

(a) an amino acid sequence of SEQ ID NO: 1; or

(b) an amino acid sequence which is at least 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 1.
The system of claim 8, further comprising one or more polynucleotides encoding a molecular chaperone, preferably the molecular chaperon is PDI or BiP, more preferably, the PDI comprises an amino acid sequence of SEQ ID NO: 10 or an amino acid sequence which is at least 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 10; and the BiP comprises an amino acid sequence of SEQ ID NO: 11 or an amino acid sequence which is at least 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 11.
A method of displaying an antibody on a host cell, comprising the steps of:

a) introducing one or more expression vector of any one of claims 1-6 into the host cell;

b) introducing one or more polynucleotide encoding a cell surface anchoring protein into the host cell; and

c) culturing the host cell under condition whereby the polypeptide encoded by expression vector from a) and cell surface anchoring protein encoded by polynucleotide from b) are expressed.
The method of claim 12, wherein the host cell is P. pastoris.
The method of claim 12, wherein the cell surface anchoring protein is ScAga1 comprises:

(a) an amino acid sequence of SEQ ID NO: 1; or

(b) an amino acid sequence which is at least 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 1.
The method of claim 12, further comprising introducing one or more polynucleotides encoding a molecular chaperone, preferably the molecular chaperon is PDI or BiP, more preferably the PDI comprises an amino acid sequencce of SEQ ID NO: 10 or an amino acid sequence which is at least 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 10; and the BiP comprises an amino acid sequence of SEQ ID NO: 11 or an amino acid sequence which is at least 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 11.